Epigenetic programming of chicken germ cells: a comparative review.

Chicken embryos serve as an important model for investigating germ cells due to their ease of accessibility and manipulation within the egg. Understanding the development of germ cells is particularly crucial, as they are the only cell types capable of transmitting genetic information to the next generation. Therefore, gene expression regulation in germ cells is important for genomic function. Epigenetic programming is a crucial biological process for the regulation of gene expression without altering the genome sequence. Although epigenetic programming is evolutionarily conserved, several differences between chickens and mammals have been revealed. In this review, we compared the epigenetic regulation of germ cells in chickens and mammals (mainly mice as a representative species). In mammals, migrating primordial germ cells (precursors for germ cells [PGCs]) undergo global DNA demethylation and persist until sexual differentiation, while in chickens, DNA is demethylated until reaching the gonad but remethylated when sexually differentiated. Prospermatogonia is methylated at the onset of mitotic arrest in mammals, while DNA is demethylated at mitotic arrest in chickens. Furthermore, genomic imprinting and inactivation of sex chromosomes are differentially regulated through DNA methylation in chickens and mammals. Chickens and mammals exhibit different patterns of histone modifications during germ cell development, and non-coding RNA, which is not involved in PGC differentiation in mice, plays an important role in chicken PGC development. Additionally, several chicken-specific non-coding RNAs have been identified. In conclusion, we summarized current knowledge of epigenetic gene regulation of chicken germ cells, comparing that of mammals, and highlighted notable differences between them.

Electrically Confined Electroluminescence of Neutral Excitons in WSe2 Light-Emitting Transistors.

Monolayer transition metal dichalcogenides (TMDs) have drawn significant attention for their potential in optoelectronic applications due to their direct band gap and exceptional quantum yield. However, TMD-based light-emitting devices have shown low external quantum efficiencies as imbalanced free carrier injection often leads to the formation of non-radiative charged excitons, limiting practical applications. Here, electrically confined electroluminescence (EL) of neutral excitons in tungsten diselenide (WSe2) light-emitting transistors (LETs) based on the van der Waals heterostructure is demonstrated. The WSe2 channel is locally doped to simultaneously inject electrons and holes to the 1D region by a local graphene gate. At balanced concentrations of injected electrons and holes, the WSe2 LETs exhibit strong EL with a high external quantum efficiency (EQE) of ≈8.2 % at room temperature. These experimental and theoretical results consistently show that the enhanced EQE could be attributed to dominant exciton emission confined at the 1D region while expelling charged excitons from the active area by precise control of external electric fields. This work shows a promising approach to enhancing the EQE of 2D light-emitting transistors and modulating the recombination of exciton complexes for excitonic devices.

Valley-centre tandem perovskite light-emitting diodes.

Perovskite light-emitting diodes (PeLEDs) have emerged as a promising new light source for displays. The development roadmap for commercializing PeLEDs should include a tandem device structure, specifically by stacking a thin nanocrystal PeLED unit and an organic light-emitting diode unit, which can achieve a vivid and efficient tandem display; however, simply combining light-emitting diodes with different characteristics does not guarantee both narrowband emission and high efficiency, as it may cause a broadened electroluminescence spectra and a charge imbalance. Here, by conducting optical simulations of the hybrid tandem (h-tandem) PeLED, we have discovered a crucial optical microcavity structure known as the h-tandem valley, which enables the h-tandem PeLED to emit light with a narrow bandwidth. Specifically, the centre structure of the h-tandem valley (we call it valley-centre tandem) demonstrates near-perfect charge balance and optimal microcavity effects. As a result, the h-tandem PeLED achieves a high external quantum efficiency of 37.0% and high colour purity with a narrow full-width at half-maximum of 27.3 nm (versus 64.5 nm in organic light-emitting diodes) along with a fast on-off response. These findings offer a new strategy to overcome the limitations of nanocrystal-based PeLEDs, providing valuable optical and electrical guidelines for integrating different types of light-emitting device into practical display applications.

Modification of surface glycan by expression of beta-1,4-N-acetyl-galactosaminyltransferase (B4GALNT2) confers resistance to multiple viruses infection in chicken fibroblast cell.

Introduction: Infectious viruses in poultry, such as avian influenza virus (AIV) and Newcastle disease virus (NDV), are one of the most major threats to the poultry industry, resulting in enormous economic losses. AIVs and NDVs preferentially recognize α-2,3-linked sialic acid to bind to target cells. The human beta-1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2) modifies α-2,3-linked sialic acid-containing glycan by transferring N-acetylgalactosamine to the sub-terminal galactose of the glycan, thus playing a pivotal role in preventing viruses from binding to cell surfaces. However, chickens lack a homolog of the B4GALNT2 gene. Methods: Here, we precisely tagged the human B4GALNT2 gene downstream of the chicken GAPDH so that the engineered cells constitutively express the human B4GALNT2. We performed a lectin binding assay to analyze the modification of α-2,3-linked sialic acid-containing glycan by human B4GALNT2. Additionally, we infected the cells with AIV and NDV and compared cell survivability, viral gene transcription, and viral titer using the WST-1 assay, RT-qPCR and TCID50 assay, respectively. Results: We validated human B4GALNT2 successfully modified α-2,3-linked sialic acid-containing glycan in chicken DF-1 cells. Following viral infection, we showed that human B4GALNT2 reduced infection of two AIV subtypes and NDV at 12-, 24-, and 36-hours post-infection. Moreover, cells expressing human B4GALNT2 showed significantly higher cell survivability compared to wild-type DF-1 cells, and viral gene expression was significantly reduced in the cells expressing human B4GALNT2. Discussion: Collectively, these results suggest that artificially expressing human B4GALNT2 in chicken is a promising strategy to acquire broad resistance against infectious viruses with a preference for α-2,3-linked sialic acids such as AIV and NDV.

Overcoming Charge Confinement in Perovskite Nanocrystal Solar Cells.

The small nanoparticle size and long-chain ligands in colloidal metal halide perovskite quantum dots (PeQDs) cause charge confinement, which impedes exciton dissociation and carrier extraction in PeQD solar cells, so they have low short-circuit current density Jsc , which impedes further increases in their power conversion efficiency (PCE). Here, a re-assembling process (RP) is developed for perovskite nanocrystalline (PeNC) films made of colloidal perovskite nanocrystals to increase Jsc in PeNC solar cells. The RP of PeNC films increases their crystallite size and eliminates long-chain ligands, and thereby overcomes the charge confinement in PeNC films. These changes facilitate exciton dissociation and increase carrier extraction in PeNC solar cells. By use of this method, the gradient-bandgap PeNC solar cells achieve a Jsc = 19.30 mA cm-2 without compromising the photovoltage, and yield a high PCE of 16.46% with negligible hysteresis and good stability. This work provides a new strategy to process PeNC films and pave the way for high performance PeNC optoelectronic devices.

Strategies for the Generation of Gene Modified Avian Models: Advancement in Avian Germline Transmission, Genome Editing, and Applications.

Avian models are valuable for studies of development and reproduction and have important implications for food production. Rapid advances in genome-editing technologies have enabled the establishment of avian species as unique agricultural, industrial, disease-resistant, and pharmaceutical models. The direct introduction of genome-editing tools, such as the clustered regularly interspaced short palindromic repeats (CRISPR) system, into early embryos has been achieved in various animal taxa. However, in birds, the introduction of the CRISPR system into primordial germ cells (PGCs), a germline-competent stem cell, is considered a much more reliable approach for the development of genome-edited models. After genome editing, PGCs are transplanted into the embryo to establish germline chimera, which are crossed to produce genome-edited birds. In addition, various methods, including delivery by liposomal and viral vectors, have been employed for gene editing in vivo. Genome-edited birds have wide applications in bio-pharmaceutical production and as models for disease resistance and biological research. In conclusion, the application of the CRISPR system to avian PGCs is an efficient approach for the production of genome-edited birds and transgenic avian models.

Single-cell transcriptome analysis of male chicken germ cells reveals changes in signaling pathway-related gene expression profiles during mitotic arrest.

Mitotic arrest is necessary for the embryonic development of germ cells, and thus, it is important to understand the signaling pathways that regulate mitotic arrest. Here, we investigated the signaling pathway dynamics of male embryonic chicken germ cells during mitotic arrest by single-cell transcriptome analysis using germ-cell tracing models. We identified signaling pathways that change at the transcriptional level during chicken male germ-cell development after sex determination. We found that several components of the BMP, Notch, and JAK-STAT signaling pathways were downregulated at the mitotic-arrest stage and were reactivated 1 week after hatching when all germ cells are quiescent after entering mitotic arrest. In addition, the transcriptional levels of components of the MAPK, Hedgehog, and thyroid-hormone signaling pathways were steadily upregulated after mitotic arrest. This suggests the cooperation of multiple signaling pathways during entry into mitotic arrest and subsequent quiescence of chicken male germ cells.

Finer resolution analysis of transcriptional programming during the active migration of chicken primordial germ cells.

Primordial germ cells (PGCs) in chickens polarize and move passively toward the anterior region by the morphogenetic movement of the embryo. Further migration of PGCs towards the genital ridge via the germinal crescent region and blood vessels occurs actively through the chemoattractive signals. The mechanisms of initiation of PGCs migration, lodging the PGCs in the vascular system, and colonization of PGCs in the gonads are well-studied. However, transcriptome sequencing-based cues directing the migration of the PGCs towards gonads, some of the relevant molecules, biological processes, and transcription factors (TFs) are less studied in chickens. The current study comprehensively interprets the transcriptional programming of PGCs during their active migration (E2.5 to E8). Current results revealed several vital understandings, including a set of genes that upregulated male-specifically (XPA, GNG10, RPL17, RPS23, and NDUFS4) or female-specifically (HINTW, NIPBL, TERAL2, ATP5F1AW, and SMAD2W) in migrating PGCs, and transcriptionally distinct PGCs, particularly in the gonadal environment. We identified DNA methylation and histone modification-associated genes that are novel in chicken PGCs and show a time-dependent enrichment in migrating PGCs. We further identified a large number of differentially expressed genes (DEGs, including TFs) in blood PGCs (at E2.5) compared to gonadal PGCs (at E8) in both sexes; however, this difference was greater in males. We also revealed the enriched biological processes and signaling pathways of significant DEGs identified commonly, male-specifically, or female-specifically between the PGCs isolated at E2.5, E6, and E8. Collectively, these analyses provide molecular insights into chicken PGCs during their active migration phase.

Ultra-bright, efficient and stable perovskite light-emitting diodes.

Metal halide perovskites are attracting a lot of attention as next-generation light-emitting materials owing to their excellent emission properties, with narrow band emission1-4. However, perovskite light-emitting diodes (PeLEDs), irrespective of their material type (polycrystals or nanocrystals), have not realized high luminance, high efficiency and long lifetime simultaneously, as they are influenced by intrinsic limitations related to the trade-off of properties between charge transport and confinement in each type of perovskite material5-8. Here, we report an ultra-bright, efficient and stable PeLED made of core/shell perovskite nanocrystals with a size of approximately 10 nm, obtained using a simple in situ reaction of benzylphosphonic acid (BPA) additive with three-dimensional (3D) polycrystalline perovskite films, without separate synthesis processes. During the reaction, large 3D crystals are split into nanocrystals and the BPA surrounds the nanocrystals, achieving strong carrier confinement. The BPA shell passivates the undercoordinated lead atoms by forming covalent bonds, and thereby greatly reduces the trap density while maintaining good charge-transport properties for the 3D perovskites. We demonstrate simultaneously efficient, bright and stable PeLEDs that have a maximum brightness of approximately 470,000 cd m-2, maximum external quantum efficiency of 28.9% (average = 25.2 ± 1.6% over 40 devices), maximum current efficiency of 151 cd A-1 and half-lifetime of 520 h at 1,000 cd m-2 (estimated half-lifetime >30,000 h at 100 cd m-2). Our work sheds light on the possibility that PeLEDs can be commercialized in the future display industry.

Dense Local Triplet States and Steric Shielding of a Multi-Resonance TADF Emitter Enable High-Performance Deep-Blue OLEDs.

Multi-resonance thermally activated delayed fluorescence (MR-TADF) molecules based on boron and nitrogen atoms are emerging as next-generation blue emitters for organic light-emitting diodes (OLEDs) due to their narrow emission spectra and triplet harvesting properties. However, intermolecular aggregation stemming from the planar structure of typical MR-TADF molecules that leads to concentration quenching and broadened spectra limits the utilization of the full potential of MR-TADF emitters. Herein, a deep-blue MR-TADF emitter, pBP-DABNA-Me, is developed to suppress intermolecular interactions effectively. Furthermore, photophysical investigation and theoretical calculations reveal that adding biphenyl moieties to the core body creates dense local triplet states in the vicinity of S1 and T1 energetically, letting the emitter harvest excitons efficiently. OLEDs based on pBP-DABNA-Me show a high external quantum efficiency (EQE) of 23.4% and a pure-blue emission with a Commission Internationale de L'Eclairage (CIE) coordinate of (0.132, 0.092), which are maintained even at a high doping concentration of 100 wt%. Furthermore, by incorporating a conventional TADF sensitizer, deep-blue OLEDs with a CIE value of (0.133, 0.109) and an extremely high EQE of 30.1% are realized. These findings provide insight into design strategies for developing efficient deep-blue MR-TADF emitters with fast triplet upconversion and suppressed self-aggregation.

Development and characterization of a CRISPR/Cas9-mediated RAG1 knockout chicken model lacking mature B and T cells.

Although birds have been used historically as a model animal for immunological research, resulting in remarkable achievements, immune cell development in birds themselves has yet to be fully elucidated. In this study, we firstly generated an immunodeficient chicken model using a CRISPR/Cas9-mediated recombination activating gene 1 (RAG1) knockout, to investigate avian-specific immune cell development. Unlike previously reported immunoglobulin (Ig) heavy chain knockout chickens, the proportion and development of B cells in both RAG1 +/- and RAG1 -/- embryos were significantly impaired during B cell proliferation (embryonic day 16 to 18). Our findings indicate that, this is likely due to disordered B cell receptor (BCR)-mediated signaling and interaction of CXC motif chemokine receptor (CXCR4) with CXCL12, resulting from disrupted Ig V(D)J recombination at the embryonic stage. Histological analysis after hatching showed that, unlike wild-type (WT) and RAG1 +/- chickens, lymphatic organs in 3-week old RAG1 -/- chickens were severely damaged. Furthermore, relative to WT chickens, RAG1+/- and RAG1-/- birds had reduced serum Igs, fewer mature CD4+ and CD8+ T lymphocytes. Furthermore, BCR-mediated B cell activation in RAG1 +/- chickens was insufficient, leading to decreased expression of the activation-induced deaminase (AID) gene, which is important for Ig gene conversion. Overall, this immunodeficient chicken model underlines the pivotal role of RAG1 in immature B cell development, Ig gene conversion during embryonic stages, and demonstrates the dose-dependent regulatory role of RAG1 during immune cell development. This model will provide ongoing insights for understanding chicken immune system development and applied in the fields of immunology and biomedical science.

Dissecting chicken germ cell dynamics by combining a germ cell tracing transgenic chicken model with single-cell RNA sequencing.

Avian germ cells can be distinguished by certain characteristics during development. On the basis of these characteristics, germ cells can be used for germline transmission. However, the dynamic transcriptional landscape of avian germ cells during development is unknown. Here, we used a novel germ-cell-tracing method to monitor and isolate chicken germ cells at different stages of development. We targeted the deleted in azoospermia like (DAZL) gene, a germ-cell-specific marker, to integrate a green fluorescent protein (GFP) reporter gene without affecting endogenous DAZL expression. The resulting transgenic chickens (DAZL::GFP) were used to uncover the dynamic transcriptional landscape of avian germ cells. Single-cell RNA sequencing of 4,752 male and 13,028 female DAZL::GFP germ cells isolated from embryonic day E2.5 to 1 week post-hatch identified sex-specific developmental stages (4 stages in male and 5 stages in female) and trajectories (apoptosis and meiosis paths in female) of chicken germ cells. The male and female trajectories were characterized by a gradual acquisition of stage-specific transcription factor activities. We also identified evolutionary conserved and species-specific gene expression programs during both chicken and human germ-cell development. Collectively, these novel analyses provide mechanistic insights into chicken germ-cell development.

Amplification of immunity by engineering chicken MDA5 combined with the C terminal domain (CTD) of RIG-I.

Innate immune system is triggered by pattern recognition receptors (PRRs) recognition. Retinoic acid-inducible gene 1 (RIG-I) is a major sensor that recognizes RNA ligands. However, chickens have no homologue of RIG-I; instead, they rely on melanoma differentiation-associated protein 5 (MDA5) to recognize RNA ligands, which renders chickens susceptible to infection by influenza A viruses (IAVs). Here, we engineered the cMDA5 viral RNA sensing domain (C-terminal domain, CTD) such that it functions similarly to human RIG-I (hRIG-I) by mutating histidine 925 into phenylalanine, a key residue for hRIG-I RNA binding loop function, or by swapping the CTD of cMDA5 with that of hRIG-I or duck RIG-I (dRIG-I). The engineered cMDA5 gene was expressed in cMDA5 knockout DF-1 cells, and interferon-beta (IFN-ß) activity and expression of interferon-related genes were measured after transfection of cells with RNA ligands of hRIG-I or human MDA5 (hMDA5). We found that both mutant cMDA5 and engineered cMDA5 triggered significantly stronger interferon-mediated immune responses than wild-type cMDA5. Moreover, engineered cMDA5 reduced the IAV titer by 100-fold compared with that in control cells. Collectively, engineered cMDA5/RIG-I CTD significantly enhanced interferon-mediated immune responses, making them invaluable strategies for production of IAV-resistant chickens. KEY POINTS: • Mutant chicken MDA5 with critical residue of RIG-I (phenylalanine) enhanced immunity. • Engineered chicken MDA5 with CTD of RIG-I increased IFN-mediated immune responses. • Engineered chicken MDA5 reduced influenza A virus titers by up to 100-fold.

Chicken blastoderms and primordial germ cells possess a higher expression of DNA repair genes and lower expression of apoptosis genes to preserve their genome stability.

DNA is susceptible to damage by various sources. When the DNA is damaged, the cell repairs the damage through an appropriate DNA repair pathway. When the cell fails to repair DNA damage, apoptosis is initiated. Although several genes are involved in five major DNA repair pathways and two major apoptosis pathways, a comprehensive understanding of those gene expression is not well-understood in chicken tissues. We performed whole-transcriptome sequencing (WTS) analysis in the chicken embryonic fibroblasts (CEFs), stage X blastoderms, and primordial germ cells (PGCs) to uncover this deficiency. Stage X blastoderms mostly consist of undifferentiated progenitor (pluripotent) cells that have the potency to differentiate into all cell types. PGCs are also undifferentiated progenitor cells that later differentiate into male and female germ cells. CEFs are differentiated and abundant somatic cells. Through WTS analysis, we identified that the DNA repair pathway genes were expressed more highly in blastoderms and high in PGCs than CEFs. Besides, the apoptosis pathway genes were expressed low in blastoderms and PGCs than CEFs. We have also examined the WTS-based expression profiling of candidate pluripotency regulating genes due to the conserved properties of blastoderms and PGCs. In the results, a limited number of pluripotency genes, especially the core transcriptional network, were detected higher in both blastoderms and PGCs than CEFs. Next, we treated the CEFs, blastoderm cells, and PGCs with hydrogen peroxide (H2O2) for 1 h to induce DNA damage. Then, the H2O2 treated cells were incubated in fresh media for 3-12 h to observe DNA repair. Subsequent analyses in treated cells found that blastoderm cells and PGCs were more likely to undergo apoptosis along with the loss of pluripotency and less likely to undergo DNA repair, contrasting with CEFs. These properties of blastoderms and PGCs should be necessary to preserve genome stability during the development of early embryos and germ cells, respectively.

Breaking the Efficiency Limit of Deep-Blue Fluorescent OLEDs Based on Anthracene Derivatives.

Triplet harvesting is important for the realization of high-efficiency fluorescent organic light-emitting diodes (OLEDs). Triplet-triplet annihilation (TTA) is one triplet-harvesting strategy. However, for blue-emitting anthracene derivatives, the theoretical maximum radiative singlet-exciton ratio generated from the TTA process is known to be 15% in addition to the initially generated singlets of 25%, which is insufficient for high-efficiency fluorescent devices. In this study, nearly 25% of the radiative singlet-exciton ratio is realized by TTA using an anthracene derivative, breaking the theoretical limit. As a result, efficient deep-blue TTA fluorescent devices are developed, exhibiting external quantum efficiencies of 10.2% and 8.6% with Commission Internationale de l'Eclairage color coordinates of (0.134, 0.131) and (0.137, 0.076), respectively. The theoretical model provided herein explains the experimental results considering both the TTA and reverse intersystem crossing to a singlet state from higher triplet states formed by the TTA, clearly demonstrating that the radiative singlet ratio generated from TTA can reach 37.5% (total radiative singlet-exciton ratio: 62.5%), well above 15% (total 40%), despite the molecule having S1 , T2  < 2T1  < Q1 energy levels, which will lead to the development of high-efficiency fluorescent OLEDs with external quantum efficiencies exceeding 28% if the outcoupling efficiency is 45%.

Asp149 and Asp152 in chicken and human ANP32A play an essential role in the interaction with influenza viral polymerase.

The acidic nuclear phosphoprotein 32 family member A (ANP32A) is a cellular host factor that determines the host tropism of the viral polymerase (vPol) of avian influenza viruses (AIVs). Compared with human ANP32A (hANP32A), chicken ANP32A contains an additional 33 amino acid residues (176-208) duplicated from amino acid residues 149-175 (27 residues), suggesting that these residues could be involved in increasing vPol activity by strengthening interactions between ANP32A and vPol. However, the molecular interactions and functional roles of the 27 residues within hANP32A during AIV vPol activity remain unclear. Here, we examined the functional role of 27 residues of hANP32A based on comparisons with other human (h) ANP32 family members. It was notable that unlike hANP32A and hANP32B, hANP32C could not support vPol activity or replication of AIVs, despite the fact that hANP32C shares a higher sequence identity with hANP32A than hANP32B. Pairwise comparison between hANP32A and hANP32C revealed that Asp149 (D149) and Asp152 (D152) are involved in hydrogen bonding and electrostatic interactions, respectively, which support vPol activity. Mutation of these residues reduced the interaction between hANP32A and vPol. Finally, we demonstrated that precise substitution of the identified residues within chicken ANP32A via homology-directed repair using the CRISPR/Cas9 system resulted in a marked reduction of viral replication in chicken cells. These results increase our understanding of ANP32A function and may facilitate the development of AIV-resistant chickens via precise modification of residues within ANP32A.

TD-DFT and Experimental Methods for Unraveling the Energy Distribution of Charge-Transfer Triplet/Singlet States of a TADF Molecule in a Frozen Matrix.

Reverse intersystem crossing (RISC) rate of a thermally activated delayed fluorescence (TADF) molecule is sensitive to the energy alignment of the singlet charge-transfer state (1CT), triplet charge-transfer state (3CT), and locally excited triplet state (3LE). However, the energy distribution of the charge-transfer states originating from the conformational distribution of TADF molecules in a solid matrix inevitably generated during the preparation of a solid sample due to the rotatable donor-acceptor linkage is rarely considered. Moreover, the investigation of the energy distribution of the 3CT state is both theoretically and experimentally difficult due to the triplet instabilities of time-dependent density functional (TD-DFT) calculations and difficulties in phosphorescence measurements, respectively. As a result, the relationships between conformational distribution, configurations of excited state transition orbitals, and excited state energies/dynamics have not been clearly explained. In this work, we determined the energy distribution of CT states of the TADF emitter TPSA in frozen toluene at 77 K by the measurement of time-resolved spectra in the full time range (1 ns to 30 s) of emission including prompt fluorescence, TADF, 3CT phosphorescence, and 3LE phosphorescence. We obtained the energy band of CT states where 1CT and 3CT states are distributed in the range of 2.85-3.00 and 2.64-2.96 eV, respectively. We tested various global hybrid and long-range corrected functionals for the TD-DFT calculation of 3CT energy of TPSA and found that only the M11 functional shows consistent results without triplet instability. We performed TD-DFT with the M11* functional optimized for a robust dihedral angle scan of 3CT states without triplet instability and reproduced the energy band structure obtained from the experiment. Through TD-DFT and experimental investigations, it is estimated that the dihedral angles of donor-acceptor (θD-A) and acceptor-linker (θA) of TPSA in frozen toluene lie within the range 70° ≤ θD-A ≤ 90° and 0° ≤ θA ≤ 30° respectively. Our results show that the dihedral angle distribution must be considered for further investigation of the photophysics of TADF molecules and the development of stable and efficient TADF emitters.

Establishment of a genetically engineered chicken DF-1 cell line for efficient amplification of influenza viruses in the absence of trypsin.

BACKGROUND: The initial step of influenza infection is binding of the virus to specific sialic acid receptors expressed by host cells. This is followed by cell entry via endocytosis. Cleavage of the influenza virus hemagglutinin (HA) protein is critical for infection; this is performed by host cell proteases during viral replication. In cell culture systems, HA is cleaved by trypsin added to the culture medium. The vast majority of established cell lines are mammalian. RESULTS: In the present study, we generated genetically engineered chicken DF-1 cell lines overexpressing transmembrane protease, serine 2 (TMPRSS2, which cleaves HA), ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3GAL1, which plays a role in synthesis of α-2,3 linked sialic acids to which avian-adapted viruses bind preferentially), or both. We found that overexpression of TMPRSS2 supports the virus life cycle by cleaving HA. Furthermore, we found that overexpression of ST3GAL1 increased the viral titer. Finally, we showed that overexpression of both TMPRSS2 and ST3GAL1 increased the final viral titer due to enhanced support of viral replication and prolonged viability of the cells. In addition, overexpression of these genes of interest had no effect on cell proliferation and viability. CONCLUSIONS: Taken together, the results indicate that these engineered cells could be used as a cell-based system to propagate influenza virus efficiently in the absence of trypsin. Further studies on influenza virus interactions with chicken cell host factors could be studied without the effect of trypsin on cells.

Highly Efficient Deep-Blue OLEDs using a TADF Emitter with a Narrow Emission Spectrum and High Horizontal Emitting Dipole Ratio.

New blue (DBA-SAB) and deep-blue (TDBA-SAF) thermally activated delayed fluorescence (TADF) emitters are synthesized for blue-emitting organic-light emitting diodes (OLEDs) by incorporating spiro-biacridine and spiro-acridine fluorene donor units with an oxygen-bridged boron acceptor unit, respectively. The molecules show blue and deep-blue emission because of the deep highest occupied molecular energy levels of the donor units. Besides, both emitters exhibit narrow emission spectra with the full-width at half maximum (FWHM) of less than 65 nm due to the rigid donor and acceptor units. In addition, the long molecular structure along the transition dipole moment direction results in a high horizontal emitting dipole ratio over 80%. By combining the effects, the OLED utilizing DBA-SAB as the emitter exhibits a maximum external quantum efficiency (EQE) of 25.7% and 1931 Commission Internationale de l'éclairage (CIE) coordinates of (0.144, 0.212). Even a higher efficiency deep blue TADF OLED with a maximum EQE of 28.2% and CIE coordinates of (0.142, 0.090) is realized using TDBA-SAF as the emitter.

Targeted Knockout of MDA5 and TLR3 in the DF-1 Chicken Fibroblast Cell Line Impairs Innate Immune Response Against RNA Ligands.

The innate immune system, which senses invading pathogens, plays a critical role as the first line of host defense. After recognition of foreign RNA ligands (e.g., RNA viruses), host cells generate an innate immune or antiviral response via the interferon-mediated signaling pathway. Retinoic acid-inducible gene I (RIG-1) acts as a major sensor that recognizes a broad range of RNA ligands in mammals; however, chickens lack a RIG-1 homolog, meaning that RNA ligands should be recognized by other cellular sensors such as melanoma differentiation-associated protein 5 (MDA5) and toll-like receptors (TLRs). However, it is unclear which of these cellular sensors compensates for the loss of RIG-1 to act as the major sensor for RNA ligands. Here, we show that chicken MDA5 (cMDA5), rather than chicken TLRs (cTLRs), plays a pivotal role in the recognition of RNA ligands, including poly I:C and influenza virus. First, we used a knockdown approach to show that both cMDA5 and cTLR3 play roles in inducing interferon-mediated innate immune responses against RNA ligands in chicken DF-1 cells. Furthermore, targeted knockout of cMDA5 or cTLR3 in chicken DF-1 cells revealed that loss of cMDA5 impaired the innate immune responses against RNA ligands; however, the responses against RNA ligands were retained after loss of cTLR3. In addition, double knockout of cMDA5 and cTLR3 in chicken DF-1 cells abolished the innate immune responses against RNA ligands, suggesting that cMDA5 is the major sensor whereas cTLR3 is a secondary sensor. Taken together, these findings provide an understanding of the functional role of cMDA5 in the recognition of RNA ligands in chicken DF-1 cells and may facilitate the development of an innate immune-deficient cell line or chicken model.